The present invention relates to glycoconjugates of 20(S)-camptothecin, in which a 3-O-methylated xcex2-L-fucose unit is linked to the 20-hydroxyl group of a camptothecin derivative via thiourea-modified peptide spacers. The invention furthermnore relates to processes for the preparation of the compounds according to the invention and to their use as medicaments, in particular in connection with oncoses.
20-(S)-Camptothecin is a pentacyclic alkaloid which was isolated in 1966 by Wall et al. (J. Am. Chem. Soc. 88, 3888 (1966)). It has a high antitumour active potential in numerous in vitro and in vivo tests. Unfortunately, however, the promising potential failed to be realized in the clinic because of toxicity and solubility problems.
By opening of the E ring lactone and formation of the sodium salt, a water-soluble compound was obtained which is in a pH-dependent equilibrium with the ring-closed form. Here too, clinical studies have been unsuccessful until now. 
Approximately 20 years later, it was found that the biological activity is to be attributed to an enzyme inhibition of the topoisomerase I. Since then, the research activities have been increased again in order to find camptothecin derivatives which are more compatible and active in vivo.
To improve the water-solubility, salts of A ring- and B ring-modified camptothecin derivatives and of 20-O-acyl derivatives having ionizable groups have been described (Vishnuvajjala et al. U.S. Pat. No. 4,943,579). The latter prodrug concept was later also applied to modified camptothecin derivatives (Wani et al. WO 9602546). In vivo, however, the 20-O-acyl prodrugs described have a very short half-life and are very rapidly cleaved to give the parent structure.
WO 9631532 A1 describes sugar-modified cytostatics in which the linkage of various cytotoxic or cytostatically active compounds to, for example, regioselectively modified carbohydrate units via specific spacers lead to an improvement in the tumour selectivity. From the combinations of carbohydrate, spacer and active compound widely described there, we then surprisingly found that the linkage of xcex2-L-fucose units modified in the 3-position via a thiourea-modified peptide spacer consisting of a sterically demanding non-polar side chain-containing and a basic side chain-containing amino acid on the 20-hydroxyl group of 20(S)-camptothecin leads to very particularly preferred conjugates having the following properties:
By means of the ester-like linkage of the carrier radical to the 20-hydroxyl group, the lactone ring in the camptothecin moiety, which is important for the action, is stabilized.
By means of the special conformation of the dipeptide spacers, the conjugates in extracellular medium and in blood have a stability which is again markedly improved in comparison with similar conjugates having other spacers previously described in WO 9631532. In particular, the conjugates according to the invention are more stable than the 20-O-acyl prodrugs of camptothecin described in U.S. Pat. No. 4,943,579.
The conjugates according to the invention have better water solubility in comparison with similar conjugates from WO 9631532.
In vitro, the conjugates according to the invention have a high activity against tumour cell lines and tumour xenografts.
In vivo, the conjugates according to the invention have excellent therapeutic activity over several dose stages against various tumours after i.v. administration.
Compared with the underlying toxophore they have a markedly higher tolerability and tumour selectivity, in particular with respect to bone marrow toxicity.
The invention relates to compounds of the formula (I) 
in which
R1 represents a sterically demanding non-polar side chain of an amino acid and
R2 represents a basic side chain of an amino acid and their salts, stereoisomers and stereoisomer mixtures.
Preferred compounds of the formula (I) are those
in which
R1 is a branched alkyl radical having up to 4 carbon atoms and
R2 is a radical of the formula xe2x80x94(CH2)nxe2x80x94R3, where 
xe2x80x83and
n is a number 1 to 4.
Particularly preferred compounds of the general formula (I) are those in which
R1 is a branched alkyl radical of the formula 
xe2x80x83and
R2 is a radical of the formula 
The camptothecin unit can be present in the 20(R) or in the 20(S) configuration or as a mixture of these two stereoisomeric forms. The 20(S) configuration is preferred.
The amino acids can occur in the L or in the D configuration or alternatively as a mixture of D and L form.
The term xe2x80x9camino acidsxe2x80x9d in particular designates the xcex1-amino acids occurring in nature, but moreover also comprises their homologues, isomers and derivatives. As an example of isomers; enantiomers may be mentioned. Derivatives can be, for example, amino acids provided with protective groups.
Amino acids having xe2x80x9csterically demandingxe2x80x9d side chains are understood as meaning those amino acids whose side chain has a branching in the xcex2- or xcex3-position; examples which may be mentioned are valine and isoleucine or leucine.
Typical examples of amino acids having non-polar side chains which may be mentioned are:
alanine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine, methionine.
Typical examples of amino acids having basic side chains which may be mentioned are:
lysine, arginine, histidine, ornithine, diaminobutyric acid.
The compounds according to the invention are preferably present in the form of their salts. In general, salts with organic or inorganic acids may be mentioned here.
The acids which can be adducted preferably include hydrohalic acids, such as, for example, hydrochloric acid and hydrobromic acid, in particular hydrochloric acid, furthermore phosphoric acid, nitric acid, sulphuric acid, mono- and bifunctional carboxylic acids and hydroxycarboxylic acids, such as, for example, acetic acid, trifluoroacetic acid, maleic acid, malonic acid, oxalic acid, gluconic acid, succinic acid, fumaric acid, tartaric acid, citric acid, salicylic acid, sorbic acid and lactic acid and also sulphonic acids, such as, for example, p-toluenesulphonic acid, 1,5-naphthalenedisulphonic acid or camphorsulphonic acid.
The glycoconjugates according to the invention can be prepared, for example, by linkage of 20(S)-camptothecin to activated carboxyl components, which for their part can be moieties of protected amino acids, peptides or carbohydrate-modified peptides.
Preferably, the synthesis of the glycoconjugate takes place sequentially, beginning with the acylation of 20(S)-camptothecin with an N-protected carboxyl-activated unit of a non-polar sterically demanding amino acid in a suitable solvent, if appropriate in the presence of a base, according to customary methods. The amino protective group is then removed selectively by means of known methods. A unit of a basic amino acid, which, if necessary, is suitably protected is then linked and subsequently, if appropriate with retention of the side chain protective group, deblocked at the xcex1-amino function. In the key step, the linkage to the carbohydrate radical is carried out by conversion of p-aminophenyl-3-O-methyl-xcex2-L-fucopyranoside into the corresponding isothiocyanate and subsequent linkage to the deblocked xcex1-amino group of the peptidyl camptothecin. Side chain protective groups which may still be present are detached and the free amino group is optionally converted into a suitable ammonium salt.
The invention thus furthermore relates to a process for the preparation of the glycoconjugates of the formula (I) 
in which
R1 represents a sterically demanding non-polar side chain of an amino acid and
R2 represents a basic side chain of an amino acid,
or of their salts, characterized in that the isothiocyanate of the formula (II) 
is reacted with the peptidyl-camptothecin, optionally bearing a protective group in the side chain, of the formula (III) 
in which
R1 has the abovementioned meaning and
R2xe2x80x2 has the meaning of the abovementioned basic radical R2, which moreover can carry a protective group customary in peptide chemistry on the basic group
to give the glycoconjugate of the formula (IV) 
in which
R1 and R2xe2x80x2 have the meanings indicated above,
the side chain amino protective group which may be present is removed according to customary methods and the compound obtained is optionally converted into the desired salt.
Another sequence of reaction steps in the synthesis of the target compound is also conceivable. Thus, according to a likewise preferred variant, the p-isothiocyanatophenyl-3-O-methyl-xcex2-L-fucoside can also be linked first with the optionally suitably protected terminal basic amino acid, and this unit can then be reacted with the free amino group of the amino acid conjugate of 20(S)-camptothecin and the non-polar, sterically demanding amino acid. Side chain protective groups which may be present are detached and the free amino group is optionally converted into a suitable ammonium salt.
The invention therefore further relates to an alternative process for the preparation of compounds of the general formula (I) or of their salts, characterized in that the isothiocyanate of the formula (II) 
is reacted with an optionally suitably protected terminal basic amino acid of the formula (V) 
in which R2xe2x80x2 represents a basic side chain of an amino acid whose basic group can be protected,
to give an amino acid conjugate of the formula (VI) 
in which R2xe2x80x2 has the meaning indicated above,
this is then reacted with amino acid conjugates of the formula (VII) 
in which R1 has the meaning indicated above,
the side chain protective group is removed and the compounds are optionally converted into a suitable salt.
Diastereomer mixtures can be formed, in particular after linkage of the first amino acid to camptothecin. Pure diastereomers of the compounds according to the invention can be prepared by the process indicated above, for example, by separating the diastereomers in a suitable manner after linkage of the first amino acid unit to the camptothecin and subsequent protective group removal. The diastereomerically pure target compound can be prepared from a diastereomerically pure intermediate compound by the route indicated above.
The diastereomer mixture of the target compound can also be separated into the individual diastereomers in a customary manner.
The reactions can be carried out under various pressure and temperature conditions, for example 0.5 to 2 bar, and xe2x88x9230 to +100xc2x0 C., in suitable solvents such as dimethylformamide (DMF), tetrahydrofuran (THF), dichloromethane, chloroform, lower alcohols, acetonitrile, dioxane, water or in mixtures of the solvents mentioned. As a rule, reactions in DMF, dichloromethane or THF/dichloromethane at room temperature and normal pressure are preferred.
For the activation of the carboxyl groups, possible coupling reagents are those known in peptide chemistry such as described, for example, in Jakubke/Jeschkeit: Aminosxc3xa4uren, Peptide, Proteine (Amino Acids, Peptides, Proteins); Verlag Chemie 1982 or Tetrahedr. Lett. 34, 6705 (1993). N-Carboxylic anhydrides, acid chlorides or mixed anhydrides, for example, are preferred.
Furthermore suitable for the activation of the carboxyl groups is the formation of adducts with carbodiumides, e.g. N,Nxe2x80x2-diethyl-, N,Nxe2x80x2-diisopropyl-, N,Nxe2x80x2-dicyclohexylcarbodiimide, N-(3-dimethylaminopropyl)-Nxe2x80x2-ethyl-carbodiumide hydrochloride, N-cyclohexyl-Nxe2x80x2-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulphonate, or carbonyl compounds such as carbonyldiimidazole, or 1,2-oxazolium compounds such as 2-ethyl-5-phenyl-1,2-oxazolium-3-sulphate or 2-tert-butyl-5-methyl-isoxazolium perchlorate, or acylamino compounds such as 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, or propanephosphonic anhydride, or isobutyl chloroformate, or benzotriazolyloxy-tris-(dimethylamino)-phosphonium hexafluorophosphate, 1-hydroxybenzotriazole esters or N-hydroxysuccinimide esters. Furthermore, the amino acid components can also be employed in the form of a Leuch"" anhydride.
Bases employed can be, for example, triethylamine, ethyl-diisopropylamine, pyridine, N,N-dimethylaminopyridine or others.
Protective groups employed for third functions of the amino acids can be the protective groups known in peptide chemistry, for example of the urethane, alkyl, acyl, ester or amide type.
Amino protective groups in the context of the invention are the customary amino protective groups used in peptide chemistry.
These preferably include: benzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyt, 4-methoxybenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl (Boc), allyloxycarbonyl, vinyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, phthaloyl, 2,2,2-trichloroethoxycarbonyl, 2,2,2-trichloro-tert-butoxycarbonyl, menthyloxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl (Fmoc), formyl, acetyl, propionyl, pivaloyl, 2-chloroacetyl, 2-bromoacetyl, 2,2,2-trifluoroacetyl, 2,2,2-trichloroacetyl, benzoyl, benzyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, phthalimido, isovaleroyl or benzyloxymethylene, 4-nitrobenzyl, 2,4-dinitrobenzyl, 4-nitrophenyl or 2-nitrophenylsulphenyl. The Fmoc group and the Boc group are particularly preferred.
Preferred carboxyl protective groups are linear or branched C1- to C4-alkyl esters.
The removal of protective groups in appropriate reaction steps can be carried out, for example, by reaction of acid or base, hydrogenolytically or reductively in another manner.
Both in vitro and in vivo, the glycoconjugates according to the invention have a surprisingly strong antitumour activity against various tumours, in particular lung, breast, pancreas, melanoma and large intestine tumours, combined with a great selectivity against non-malignant cells.
They are therefore suitable for the treatment of oncoses, to be specific both in human and in veterinary medicine.
The present invention includes pharmaceutical preparations which, in addition to non-toxic, inert pharmaceutically suitable excipients, contain one or more compounds according to the invention or which consist of one or more active compounds according to the invention, and processes for the production of these preparations.
The active compounds can optionally be present in one or more of the excipients indicated above and also in microencapsulated form.
The therapeutically active compounds should be present in the abovementioned pharmaceutical preparations in a concentration from approximately 0.1 to 99.5, preferably from approximately 0.5 to 95%, by weight of the total mixture.
Apart from the compound according to the invention, the abovementioned pharmaceutical preparations can also contain further pharmaceutical active compounds.
In general, it has proven advantageous both in human and in veterinary medicine to administer the active compound according to the invention in total amounts of approximately 0.5 to approximately 500, preferably 5 to 100, mg/kg of body weight every 24 hours, if appropriate in the form of several individual doses, to achieve the desired results. An individual dose contains the active compound(s) according to the invention preferably in amounts from approximately 1 to 80, in particular 3 to 30, mg/kg of body weight.
1. Growth Inhibition Test for the Determination of the Cytotoxic Properties:
The human large intestine cell lines SW 480 and HT 29 (ATCC No. CCL 228 and HBT 38) and the mouse melanoma cell line B16F10 (CRL 6475) were grown in Roux dishes in RPMI 1640 medium with addition of 10% FCS. They were then trypsinized and taken up in RPMI plus 10% FCS to a cell count of 50,000 cells/ml for SW 480 and HT 29 and 20,000 cells for B16F10. 100 xcexcl of cell suspension/well were added to a 96 microwell plate and incubated for 1 day at 37xc2x0 C. in a CO2 incubator. A further 100 xcexcl of RPMI medium and 1 xcexcl of DMSO containing the test substances were then added. The growth was checked after day 6. To do this, 25 xcexcl of MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) having a starting concentration of 5 mg/ml of H2O were added to each microwell. Incubation was carried out for 5 hours in a CO2 incubator at 37xc2x0 C. The medium was then aspirated and 100 xcexcl of i-propanol/well were added. After shaking for 30 min with 100 xcexcl of H2O, the extinction was measured at 595 nm using a multiplate reader (Bio.) 3550 UV.
The cytotoxic action is indicated in Table 1 as the IC50 value in each case for the SW 480 and HT 29 and B16F10 cell lines:
2. Haematopoetic Activity of the Glycoconjugate in Comparison with the Underlying Active Compound:
Material and Methods:
Bone marrow cells were washed out of mice femurs. 105 cells were incubated at 37xc2x0 C. and 7% CO2 in McCoy 5A medium (0.3% agar) together with recombinant murine GM-CSF (Genzyme; stem cell colony formation) and the substances (10xe2x88x924 to 100 xcexcg/ml). 7 days later, the colonies ( less than 50 cells) and clusters (17-50 cells) were counted.
Results:
As shown in Tab. 2, the glycoconjugates investigated show a drastically reduced inhibition of the bone marrow stem cell proliferation compared with the underlying active compound.
3. In Vivo Inhibition of Tumour Growth in the Nude Mouse Model
Material:
For all in vivo experiments for investigation of the inhibition of tumour growth, athymic nude mice (NMRI nu/nu strain) were used. The selected large-cell lung carcinoma LXFL 529 was grown by serial passage in nude mice. The human origin of the tumour was confirmed by isoenzymatic and immunohistochemical methods.
Experimental Set-up:
The tumour was implanted subcutaneously into both flanks of small nu/nu nude mice 6 to 8 weeks old. The treatment was started, depending on the doubling time, as soon as the tumours had reached a diameter of 5-7 mm. The mice were assigned to the treatment group and the control group (5 mice per group with 8-10 assessable tumours) by randomization. The individual tumours of the control group all grew progressively.
The size of the tumours was measured in two dimensions by means of a slide gauge. The tumour volume, which correlated well with the cell count, was then used for all evaluations. The volume was calculated according to the formula xe2x80x9clengthxc3x97breadthxc3x97breadth/2xe2x80x9d ([axc3x97b2]/2, a and b represent two diameters at right angles).
The values of the relative tumour volume (RTV) were calculated for each individual tumour by dividing the tumour size on day X with the tumour size on day 0 (at the time of randomization). The mean values of the RTV were then used for the further evaluation.
The inhibition of the tumour volume (relative tumour volume of the test group/control groupxc3x97100, T/C in %) was the final measured value.
Treatment:
The administration of the compounds was carried out intravenously (i.v.), for example on day 0, 1 and 2 after randomization, the total dose per day being split over 2 administrations.
Results:
The therapeutic efficacy of the glycoconjugates according to the invention from Examples 1 and 2 is shown by way of example in the large-cell human lung tumour xenograft LXFL 529. In the case of the maximum tolerable dose (MTD) and at xc2xd MTD, the therapy leads to complete to marked tumour remission. An excellent action can also be demonstrated on other tumours.
The long-lasting complete remission of the compound from Example 1 in the dose range from 16 to 8 mg/kg and the dose-dependence of the action is shown in a further experiment in FIG. 1 using the therapy schedule day 1-3 i.v.
4. Hydrolytic Stability:
The compounds according to the invention from Examples 1, 2, 8 and 9 are dissolved in water and, after standing at room temperature for 24 h, show markedly less than 1% camptothecin release in the HPLC according to area percent.
On dissolving 10 xcexcM of the compounds from Examples 1 and 2 in RPMI medium plus 10% FCS and in 30% strength human whole blood in PBS buffer, only a camptothecin release of less than 5% took place after standing for 24 h.
Method:
HPLC system Hewlett Packard HP 1050
Column: Nucleosil 120-5 C 18 250 mmxc3x974 mm (Macherey and Nagel; Germany)
Eluent: A: 0.01 M KH2PO4 in H2O (H2=Milli-pore grade)
B: 80% acetonitrile/20% eluent A
Flow rate: 1.2 ml
Gradient: t0: 20% B-t40: 100% B
t45: 100% B-t47: 20% B
Detection: 240 nm or 370 nm
5. Lactone Stabilization:
The glycoconjugates according to the invention from Examples 1, 2 8 and 9 are dissolved in 80% water and 20% of acetonitrile and adjusted to pH 9 using 2 equivalents of sodium hydroxide solution. After standing at room temperature for 1 h, the lactone ring opening is less than 5% (detection according to the above method).